This is a vertically integrated project that will advance the emerging technology of laser scanning cytometry (LSC) to the point that it will a) enable the performance of clinically relevant tissue profiling studies consisting of 50-100 fluorescent and immunofluorescent measurements per sample in human solid tumors, grouped in panels of 5-10 correlated measurements per cell on each of approximately 5,000 cells per panel, and, b) enable extensive analysis of the data, using hypothesis-testing and/or exploratory approaches. Starting with a commercially available instrument (CompuCyte Corp., Cambridge, MA) and multicolor protocols previously developed for lymphoid tissues, during the R-21 phase we will a) develop cell preparatory methods that are suitable for epithelial tumors, b) identify the best initial measurements for identifying and contouring individual cells for multiparameter analysis (light scatter vs cytokeratin, or tubulin), c) develop one or more dye combinations to serve as templates for subsequent development of additional multicolor panels, each encompassing 4-6 correlated measurements per cell, and d) determine the conditions under which individual cells can be revisited and restained with up to 5 additional fluorescent probes per cell. During the R-33 phase we will a) develop a core set of 5-10 multicolor immunofluorescent panels for tissue profiling, each consisting of 5-10 measurements per cell with restaining, using antibodies that have been optimized with respect to non-crossreactivity and binding affinity, b) develop software that will have the capability to capture, preprocess, display, analyze, store, and export mixed data sets consisting of mixtures of correlated, partially correlated, and uncorrelated data, c) extend the capabilities of the CompuCyte instrument by adding additional lasers, and doing custom dye development to increase the number of potential correlated measurements per cell, and d) expand interactions with clinical departments within our institution, in anticipation of devising specific translational clinical studies to explore tissue profiling for prognosis and treatment planning, and launching such studies by the time this grant period has been completed.